CN117608065A

CN117608065A - Image acquisition device, image acquisition method, and spatial light modulation unit

Info

Publication number: CN117608065A
Application number: CN202311631953.3A
Authority: CN
Inventors: 泷口优
Original assignee: Hamamatsu Photonics KK
Current assignee: Hamamatsu Photonics KK
Priority date: 2015-11-06
Filing date: 2016-09-20
Publication date: 2024-02-27
Also published as: EP3373061B1; JP6570427B2; US10466458B2; CN108351502A; US20190137744A1; WO2017077777A1; JP2017090590A; EP3373061A4; EP3373061A1

Abstract

The image acquisition device (1) is provided with: a light source (11); a spatial light modulator (14) that has a plurality of pixels arranged in two dimensions and modulates the phase of excitation light (L1) output from the light source (11) for each of the plurality of pixels; a1 st objective lens (16); a2 nd objective lens (17); a photodetector (20); a control unit (21) controls the phase modulation amount of each of the plurality of pixels in accordance with a two-dimensional phase pattern (P) corresponding to the plurality of pixels. The phase pattern (P) is a phase pattern generated using a predetermined basic phase pattern (31). The basic phase pattern (31) has a1 st region (32) in which the phase value continuously increases along a prescribed direction (D1), and a2 nd region (33) which is opposite to the 1 st region (32) in the direction (D1) and in which the phase value continuously decreases along the direction (D1).

Description

Image acquisition device, image acquisition method, and spatial light modulation unit

The application is filing date2016, 9 and 20 daysApplication number is201680063190.9The invention is named asImage acquisition Acquisition device, image acquisition method, and spatial light modulation unitIs a divisional application of the patent application of (2).

Technical Field

One aspect of the present invention relates to an image acquisition device, an image acquisition method, and a spatial light modulation unit that acquire an image by capturing detection light emitted from a sample in response to irradiation of excitation light.

Background

As such an image acquisition device, there is a light sheet (light sheet) microscope that irradiates excitation light in the form of a sheet (sheet) onto a sample and picks up an image of detection light emitted from the sample in accordance with the irradiation of the excitation light (for example, refer to the following non-patent document 1). In the light sheet microscope described in non-patent document 1, a Bessel beam (Bessel beam) is generated by a spatial light modulator (SLM: spatial Light Modulator), and the focal point of the generated Bessel beam is scanned along the optical axis thereof, whereby a sheet-like excitation light is pseudo-produced.

Prior art literature

Non-patent literature

Non-patent document 1: florian O.Fahrbach and AlexanderRohrbach, "A line Scanned light-sheet microscope withphase shaped self-reconstructing beams", november 2010/Vol.18, no.23/OPTICS EXPRESS pp.24229-24244

Disclosure of Invention

Technical problem to be solved by the invention

In the light sheet microscope described in the non-patent document 1, since the condensed point of the bessel beam is scanned along the optical axis to generate the excitation light in the form of a sheet in a suspected manner, an optical element or the like for the scanning is necessary. Therefore, there is a fear that the device structure may be complicated.

An object of one aspect of the present invention is to provide an image acquisition device, an image acquisition method, and a spatial light modulation unit that can generate sheet-like excitation light with a simple configuration.

Technical means for solving the problems

An image acquisition device according to an aspect of the present invention includes: a light source that outputs excitation light including a wavelength at which a sample is excited; a spatial light modulator having a plurality of pixels arranged in two dimensions and modulating a phase of excitation light output from a light source for each of the plurality of pixels; a1 st objective lens for irradiating the sample with the excitation light modulated by the spatial light modulator; a2 nd objective lens for guiding the detection light emitted from the sample in response to the irradiation of the excitation light from the 1 st objective lens; a photodetector for capturing the detection light guided by the 2 nd objective lens; a control unit that controls the amount of phase modulation for each of the plurality of pixels in accordance with a phase pattern in which phase values corresponding to the plurality of pixels are distributed two-dimensionally; the phase pattern is a phase pattern generated based on a predetermined basic phase pattern, and the basic phase pattern includes a1 st region in which the phase value continuously increases in a predetermined direction, and a2 nd region which faces the 1 st region in the predetermined direction and in which the phase value continuously decreases in the predetermined direction.

In this image acquisition device, a phase pattern is calculated from a basic phase pattern having a1 st region in which the phase value continuously increases in a predetermined direction and a2 nd region in which the phase value continuously decreases in the predetermined direction. By modulating the excitation light with the spatial light modulator in accordance with the phase pattern, the excitation light in the form of a sheet can be irradiated from the 1 st objective lens to the sample. Therefore, it is not necessary to scan the focal point of the bessel beam in order to generate the sheet-like excitation light as in the conventional technique, and an optical element or the like for the scanning can be omitted. Therefore, according to this image acquisition device, excitation light in a sheet form can be generated with a simple configuration.

In the image acquisition apparatus according to one aspect of the present invention, the phase value may be linearly increased in the 1 st region along a predetermined direction, and the phase value may be linearly decreased in the 2 nd region along the predetermined direction. Thus, since the basic phase pattern is single-purified, the sheet-like excitation light can be generated with a simpler structure without using a complicated optical element or the like.

In the image acquisition apparatus according to one aspect of the present invention, the basic phase pattern may be line-symmetrical with respect to a straight line passing through a center in a predetermined direction and orthogonal to the predetermined direction. This can generate sheet-like excitation light on the optical axis of the 1 st objective lens. Therefore, the optical axes of the 1 st objective lens and the 2 nd objective lens can be easily adjusted.

In the image acquisition apparatus according to one aspect of the present invention, the basic phase pattern may be non-linear symmetrical with respect to a straight line passing through the center in the predetermined direction and orthogonal to the predetermined direction. This makes it possible to generate the sheet-like excitation light at a position different from the optical axis of the 1 st objective lens.

In the image pickup apparatus according to one aspect of the present invention, the 1 st region and the 2 nd region may be adjacent to each other and have a continuous phase value at a boundary thereof. Thus, since the basic phase pattern is single-purified, excitation light in a sheet form can be generated with a simpler structure without using a complicated optical element or the like.

In the image acquisition device according to one aspect of the present invention, the phase pattern may be a phase pattern in which a diffraction grating pattern and a basic phase pattern are superimposed. Thus, the phase of the excitation light can be set to a diffraction grating shape without providing a diffraction grating. Therefore, the sheet-like excitation light can be generated with a simpler structure.

In the image acquisition device according to one aspect of the present invention, the phase pattern may be a phase pattern in which a lenticular lens pattern and a basic phase pattern are superimposed. Thus, the phase of the excitation light can be set to be lenticular without providing a lens element. Therefore, the sheet-like excitation light can be generated with a simpler structure.

The image acquisition device according to one aspect of the present invention may further include an optical scanning unit that scans the excitation light with respect to the sample.

In the image acquisition device according to one aspect of the present invention, the photodetector may be a two-dimensional image pickup device having a plurality of pixel rows and capable of rolling readout. This can improve S/N compared with the case of using a two-dimensional image pickup device capable of full-scale (global) reading.

An image acquisition method according to an aspect of the present invention includes: step 1, modulating, by a spatial light modulator having a plurality of pixels arranged in two dimensions, a phase of excitation light including a wavelength at which a sample is excited, for each of the plurality of pixels; step 2, irradiating the sample with excitation light modulated by the spatial light modulator; step 3, guiding the detection light emitted from the sample along with the irradiation of the excitation light, and capturing an image of the guided detection light; in step 1, the phase modulation amount of each of the plurality of pixels is modulated in accordance with a phase pattern that is generated based on a prescribed basic phase pattern and that is two-dimensionally distributed corresponding to the phase values of the plurality of pixels, respectively, the basic phase pattern having a1 st region in which the phase values continuously increase in the prescribed direction, a2 nd region that faces the 1 st region in the prescribed direction and in which the phase values continuously decrease in the prescribed direction.

In this image acquisition method, a phase pattern is calculated from a basic phase pattern having a1 st region in which a phase value continuously increases in a predetermined direction and a2 nd region in which a phase value continuously decreases in a predetermined direction. By modulating the excitation light with the spatial light modulator in accordance with the phase pattern, the excitation light in the form of a sheet can be irradiated onto the sample. Therefore, it is not necessary to scan the focal point of the bessel beam in order to generate the sheet-like excitation light as in the conventional technique, and an optical element or the like for the scanning can be omitted. Therefore, according to this image acquisition method, excitation light in a sheet form can be generated with a simple structure.

A spatial light modulation unit according to an aspect of the present invention is a spatial light modulation unit used for a light sheet microscope, comprising: a spatial light modulator having a plurality of pixels arranged in two dimensions and modulating a phase of light input to each of the plurality of pixels and outputting the modulated light; a control unit that controls the amount of phase modulation for each of the plurality of pixels in accordance with a phase pattern in which phase values corresponding to the plurality of pixels are distributed two-dimensionally; the phase pattern is a phase pattern generated based on a predetermined basic phase pattern, and the basic phase pattern includes a1 st region in which the phase value continuously increases in a predetermined direction, and a2 nd region which faces the 1 st region in the predetermined direction and in which the phase value continuously decreases in the predetermined direction.

In the spatial light modulation unit, a phase pattern is calculated from a basic phase pattern having a1 st region in which a phase value continuously increases in a predetermined direction and a2 nd region in which a phase value continuously decreases in a predetermined direction. By modulating the excitation light with the spatial light modulator in accordance with the phase pattern, the excitation light in the form of a sheet can be irradiated onto the sample. Therefore, it is not necessary to scan the focal point of the bessel beam in order to generate the sheet-like excitation light as in the conventional technique, and an optical element or the like for the scanning can be omitted. Therefore, according to the spatial light modulation unit, excitation light in a sheet shape can be generated with a simple structure.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, excitation light in the form of a sheet can be generated with a simple structure.

Drawings

Fig. 1 is a block diagram showing a configuration of a light sheet microscope as an embodiment of an image acquisition device of the present invention.

Fig. 2 (a) is a view showing a light receiving surface of the photodetector of fig. 1, and fig. 2 (b) is a view showing rolling readout in the photodetector.

Fig. 3 is a diagram showing a basic phase pattern used in the light sheet microscope of fig. 1.

Fig. 4 is a conceptual diagram illustrating a case where sheet-like excitation light is generated.

Fig. 5 is a diagram showing a1 st modification of the basic phase pattern.

Fig. 6 is a conceptual diagram illustrating a case where excitation light of a sheet shape is generated using the basic phase pattern of fig. 5.

Fig. 7 is a diagram showing the 2 nd to 4 th modifications of the basic phase pattern.

Fig. 8 is a diagram illustrating a case where the diffraction grating pattern is superimposed on the basic phase pattern.

Fig. 9 is a diagram illustrating a case where a lens pattern is superimposed on a basic phase pattern.

Fig. 10 is a block diagram showing the structure of a light sheet microscope according to modification 5.

Fig. 11 is a conceptual diagram illustrating the case of generating excitation light in a sheet form in the light sheet microscope of fig. 10.

Detailed Description

Hereinafter, embodiments of an image acquisition apparatus and an image acquisition method according to the present invention will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals are given to the same or corresponding elements, and overlapping description is omitted.

A light sheet microscope (image acquisition device) 1 shown in fig. 1 is a device that irradiates a sample S with sheet-like excitation light L1, and captures an image of the sample S by capturing detection light L2 emitted from the sample S in response to the irradiation of the excitation light L1. In the light sheet microscope 1, the condensed position of the excitation light L1 is scanned with respect to the sample S in a direction orthogonal to the optical axis of the excitation light L1, and an image of the sample S is acquired at each condensed position. In the light sheet microscope 1, since the area where the excitation light L1 is irradiated to the sample S is narrow, degradation of the sample S such as photofading or phototoxicity can be suppressed, and image acquisition can be speeded up.

The sample S to be observed is, for example, a sample of a cell or a organism containing a fluorescent substance such as a fluorescent dye or a fluorescent gene. When light of a predetermined wavelength range is irradiated to the sample S, detection light L2 such as fluorescence is emitted. The sample S is accommodated in a holder having at least permeability to the excitation light L1 and the detection light L2, for example. The holder is held, for example, on a platform.

As shown in fig. 1, the light sheet microscope 1 includes a light source 11, a collimator lens 12, a light scanning unit 13, an SLM14, a1 st optical system 15, a1 st objective lens 16, a2 nd objective lens 17, a filter 18, a2 nd optical system 19, a photodetector 20, and a control unit 21.

The light source 11 outputs excitation light L1 including a wavelength at which the sample S is excited. The light source 11 emits, for example, coherent light or incoherent light. Examples of the coherent light source include a laser light source such as a Laser Diode (LD). Examples of the incoherent light source include a Light Emitting Diode (LED), a superluminescent diode (SLD), and a lamp-type light source. As the laser light source, a light source that oscillates a continuous wave (continuous wave) is preferable, and a light source that oscillates a pulse light such as an ultrashort pulse light may be used. As a light source for oscillating the pulsed light, a unit that combines a light source outputting the pulsed light and an optical shutter or an AOM (Acousto-optic modulator) for pulse modulation may be used. The light source 11 may be configured to output excitation light L1 including a plurality of wavelength regions. In this case, a part of the wavelength of the excitation light L1 may be selectively transmitted by an optical filter such as an acousto-optic tunable filter (acousto-optic tunable filter).

The collimator lens 12 collimates the excitation light L1 output from the light source 11 and outputs the collimated excitation light L1. The light scanning unit 13 is an optical scanner that scans the excitation light L1 with respect to the sample S by changing the traveling direction of the excitation light L1 output from the collimator lens 12. Thus, the irradiation position of the excitation light L1 irradiated to the sample S through the 1 st optical system 15 and the 1 st objective lens 16 is scanned on the surface of the sample S in a direction orthogonal to the optical axis of the 1 st objective lens 16 (the optical axis of the excitation light L1). The optical scanning unit 13 is, for example, an acousto-optic element such as a galvanometer mirror (galvano mirror), a resonance scanner, a polygon mirror, a MEMS (Micro Electro Mechanical System) mirror, an AOM, an AOD (acousto-optic deflector), or the like.

The SLM14 is a phase modulation type spatial light modulator having a plurality of pixels arranged in two dimensions and modulating the phase of the excitation light L1 output from the light source 11 for each of the plurality of pixels. The SLM14 modulates the excitation light L1 incident from the light scanning section 13 and outputs the modulated excitation light L1 toward the 1 st optical system 15 (1 st step, modulation step). The SLM14 is constituted, for example, as a transmissive type or a reflective type. Fig. 1 shows a transmissive SLM14. The SLM14 is, for example, a refractive index change material type SLM (for example, LCOS (liquid crystal on silicon (liquid crystal on silicon)) type SLM, LCD (liquid crystal display (liquid crystal display))), a variable mirror type SLM (for example, segment mirror type SLM, continuous deformable mirror (continuously deformable mirror) type SLM), or an SLM using an electrically-addressed liquid crystal element or an optically-addressed liquid crystal element, or the like. The SLM14 is electrically connected to a controller 23 of the control section 21, and constitutes a spatial light modulation unit. The driving of the SLM14 is controlled by a controller 23. Details regarding the control of the SLM14 by the control section 21 will be described later.

The 1 st optical system 15 optically combines the SLM14 and the 1 st objective lens 16 in such a manner that the excitation light L1 output from the SLM14 is guided to the 1 st objective lens 16. The 1 st optical system 15 here has a lens 15a for condensing the excitation light L1 from the SLM14 at the pupil of the 1 st objective lens 16, and constitutes a two-sided telecentric optical system.

The 1 st objective lens 16 is an objective lens for illumination, and irradiates the sample S with the excitation light L1 modulated by the SLM14 (step 2, irradiation step). The 1 st objective lens 16 is movable along its optical axis by a driving element such as a piezoelectric (piezo) actuator or a stepping motor. This can adjust the light condensing position of the excitation light L1. The 1 st optical system 15 and the 1 st objective lens 16 constitute an irradiation optical system.

The 2 nd objective lens 17 is an objective lens for detection, and guides the detection light L2 emitted from the sample S in response to the irradiation of the excitation light L1 from the 1 st objective lens 16 to the photodetector 20 side. In this example, the 2 nd objective lens 17 is disposed such that its optical axis (optical axis of the detection light L2) is orthogonal (intersects) with the optical axis of the 1 st objective lens 16. The 2 nd objective lens 17 is movable along its optical axis by a driving element such as a piezoelectric actuator or a stepping motor. Thereby, the focal position of the 2 nd objective lens 17 can be adjusted.

The filter 18 is an optical filter for separating the excitation light L1 and the detection light L2 from the light guided by the 2 nd objective lens 17 and outputting the extracted detection light L2 to the photodetector 20 side. The filter 18 is disposed on the optical path between the 2 nd objective lens 17 and the photodetector 20. The 2 nd optical system 19 optically couples the 2 nd objective lens 17 and the photodetector 20 so that the detection light L2 output from the 2 nd objective lens 17 is guided to the photodetector 20. The 2 nd optical system 19 includes a lens 19a for imaging the detection light L2 from the 2 nd objective lens 17 on the light receiving surface 20a (fig. 2) of the photodetector 20. The detection optical system is constituted by the 2 nd optical system 19 and the 2 nd objective lens 17.

The photodetector 20 captures an image of the detection light L2 guided by the 2 nd objective lens 17 and imaged on the light receiving surface 20a (step 3, imaging step). The photodetector 20 is a two-dimensional image pickup element having a plurality of pixel columns and capable of performing rolling readout for each of the plurality of pixel columns. Examples of such a photodetector 20 include a CMOS image sensor. As shown in fig. 2 (a), a pixel row R in which a plurality of pixels are arranged in a direction perpendicular to the readout direction is arranged in a plurality of rows in the readout direction on the light receiving surface 20a of the photodetector 20.

In the photodetector 20, as shown in fig. 2 (b), exposure and readout are controlled for each pixel column R by inputting a reset signal and a readout start signal according to a drive period of a drive clock. In the scroll readout, a readout start signal is sequentially input to each pixel column R at a prescribed time difference. Therefore, unlike the entire readout in which readout of all pixel columns is performed simultaneously, readout of each pixel column R is performed sequentially with a prescribed time difference.

The control unit 21 is composed of a computer 22 including a processor, a memory, and the like, and a controller 23 including a processor, a memory, and the like. The computer 22 is, for example, a personal computer or an intelligent device, and the operations of the optical scanning unit 13, the 1 st objective lens 16, the 2 nd objective lens 17, the photodetector 20, the controller 23, and the like are controlled by a processor to perform various controls. For example, the computer 22 performs control for synchronizing scanning of the excitation light L1 by the light scanning unit 13 with timing of image capturing of the detection light L2 by the photodetector 20. Specifically, the detection light L2 detected on the photodetector 20 is also moved in response to the scanning of the excitation light L1 by the light scanning unit 13. Therefore, the computer 22 controls the photodetector 20 or the optical scanning unit 13 so that signal readout by the rolling readout is performed in accordance with the movement of the detection light L2 on the photodetector 20.

The controller 23 is electrically connected to the computer 22 and controls the amount of phase modulation of each of the plurality of pixels on the SLM14 in accordance with a two-dimensional phase pattern P as shown in fig. 3. The phase pattern P is a pattern of phase values related to positions on a two-dimensional plane, each position in the phase pattern P corresponding to a plurality of pixels of the SLM14. The phase value of the phase pattern P is specified to be between 0 and 2 pi radians. In fig. 3, the phase values on each part of the phase pattern P are represented by color density. The upper limit of the phase value of the phase pattern P may be larger than 2pi radians.

The controller 23 controls the phase modulation amount of each pixel of the SLM14 for the pixel in accordance with the phase value of the position corresponding to the pixel in the phase pattern P. Specifically, for example, a D/a conversion section (digital/analog converter) that converts a phase value of a phase pattern P, which is input as digital data such as DVI (digital video interface (digital video interface)), into a drive voltage value applied to each pixel is provided in the controller 23. The controller 23 converts the phase value of the phase pattern P into a driving voltage value by the D/a conversion section if the phase pattern P is input to the controller 23 from the computer 22, and inputs the driving voltage value to the SLM14. The SLM14 applies voltages to the respective pixels corresponding to the inputted drive voltage values. Also, for example, the SLM14 has a D/a conversion section, and the controller 23 can also input digital data corresponding to the phase pattern P to the SLM14. In this case, the phase value of the phase pattern P is converted into a drive voltage value by the D/a conversion section of the SLM14. In addition, the SLM14 may control the voltage value applied to each pixel according to the digital signal output from the controller 22 without performing the D/a conversion.

The phase pattern P is calculated by the computer 22 of the control unit 21 from the predetermined basic phase pattern 31. The basic phase pattern 31 is stored in advance in the memory of the computer 22, for example. By modulating the excitation light L1 with the SLM14 in accordance with the phase pattern P calculated from the basic phase pattern 31, the sheet-like excitation light L1 can be irradiated from the 1 st objective lens 16. As will be described later, the phase pattern P may be calculated by further superimposing another pattern on the basic phase pattern 31, and the basic phase pattern 31 will be used as the phase pattern P.

As shown in fig. 3, the basic phase pattern 31 is set within a rectangular range. The basic phase pattern 31 includes a rectangular 1 st region 32 in which the phase value continuously increases along a predetermined direction D1, and a rectangular 2 nd region 33 in which the phase value continuously decreases along the direction D1 while being opposed to the 1 st region 32 in the direction D1. That is, in the 1 st region 32 and the 2 nd region 33, the directions in which the phase values are increased and decreased are opposite to each other. In a certain area, the term "the phase value continuously increases" means that the phase value continuously extends over the entire area. The phase value of 0 radian and the phase value of 2pi radian are the same, and the phase value is continuous even if the phase value varies between 0 radian and 2pi radian.

In the 1 st region 32, the phase value increases straight along the direction D1. In the 2 nd region 33, the phase value decreases straight along the direction D1. Even in either of the 1 st region 32 and the 2 nd region 33, the phase value changes by only 2π radians. That is, the absolute value of the slope (the proportion of increase) of the phase value in the 1 st region 32 and the absolute value of the slope (the proportion of decrease) of the phase value in the 2 nd region 33 are equal. Even in any of the 1 st region 32 and the 2 nd region 33, the phase value becomes constant along the direction D2 orthogonal to the direction D1. The 1 st region 32 and the 2 nd region 33 are adjacent to each other, and the phase values are continuous at the boundaries thereof. In this example, the phase value on the boundary becomes 0 radian. The basic phase pattern 31 is line-symmetrical about a straight line (center line) C passing through the center in the direction D1 and orthogonal to the direction D1. In this example, the boundary of the 1 st region 32 and the 2 nd region 33 is located on the center line C.

Fig. 4 is a conceptual diagram illustrating a case where a sheet-like excitation light is generated by modulating the excitation light L1 with the SLM14 in accordance with the basic phase pattern 31. Fig. 4 (a) is a diagram showing the optical path of the excitation light L1 when viewed from the direction D2 corresponding to the direction D2, and fig. 4 (b) is a diagram showing the optical path of the excitation light L1 when viewed from the direction D1 corresponding to the direction D1. In fig. 4 (a), as an example of the optical path of the excitation light L1 incident on the 1 st region 32, 3 excitation lights A1, B1, C1 are shown in order of decreasing distance from the optical axis X of the 1 st objective lens 16. As an example of the optical path of the excitation light L1 incident on the 2 nd region 33, 3 excitation lights A2, B2, and C2 are shown in order of decreasing distance from the optical axis X.

As shown in fig. 4 (a), the excitation light A1, A2 is delayed in phase by a prescribed amount on the SLM14 and imaged on the optical axis X. In the excitation light B1, B2, the amount of retardation of the phase on the SLM14 is larger than that of the excitation light A1, A2. Therefore, the excitation light B1, B2 is imaged on the optical axis X farther from the position of the 1 st objective lens 16 than the excitation light A1, A2. In the excitation light C1, C2, the retardation of the phase on the SLM14 is smaller than that of the excitation light A1, A2, and the phase is not substantially changed on the SLM14. Therefore, the excitation light C1, C2 is imaged on the optical axis X at a position closer to the 1 st objective lens 16 than the excitation light A1, A2.

As shown in fig. 4 (b), the phase of the excitation light L1 does not change on the SLM14 when viewed from the direction d 1. In summary, the sheet-like excitation light L1 is generated at the predetermined imaging position Y1. In this example, the sheet-like excitation light L1 is generated on the optical axis X such that the width direction thereof is along the direction d 2.

As described above, in the light sheet microscope 1, the phase pattern P is calculated from the basic phase pattern 31 having the 1 st region 32 in which the phase value continuously increases along the direction D1 and the 2 nd region 33 in which the phase value continuously decreases along the direction D1. By modulating the excitation light with the SLM14 in accordance with the phase pattern P, the 1 st objective lens 16 can irradiate the excitation light L1 in a sheet form to the sample S. Therefore, it is not necessary to scan the converging point of the bessel beam in order to generate the sheet-like excitation light L1 as in the conventional technique, and an optical element or the like for the scanning can be omitted. Therefore, according to the light sheet microscope 1, the sheet-like excitation light L1 can be generated with a simple structure. Further, since it is not necessary to scan the converging point of the bessel beam in order to generate the sheet-like excitation light L1 as in the conventional technique, control can be facilitated and the time required for image acquisition can be shortened.

In the light sheet microscope 1, the phase value linearly increases in the 1 st region 32 along the direction D1, and the phase value linearly decreases in the 2 nd region 33 along the direction D1. Thus, since the basic phase pattern 31 is single-purified, the sheet-like excitation light L1 can be generated with a simpler structure without using a complicated optical element or the like. That is, the phase value does not linearly increase along the direction D1 in at least one of the 1 st region 32 and the 2 nd region 33, and in this case, there is a fear that the configuration of the 1 st optical system 15, the 1 st objective lens 16, or the like for generating the sheet-like excitation light L1 may be complicated. In contrast, in the light sheet microscope 1, the phase value increases linearly along the direction D1 even in any of the 1 st region 32 and the 2 nd region 33, so that the configuration of the optical system for generating the sheet-like excitation light L1 can be simplified.

In the light sheet microscope 1, the basic phase pattern 31 is line-symmetrical with respect to the straight line C. This enables the sheet-like excitation light L1 to be generated on the optical axis X of the 1 st objective lens 16. Therefore, the optical axes of the 1 st objective lens 16 and the 2 nd objective lens 17 can be easily adjusted.

In the light sheet microscope 1, the 1 st region 32 and the 2 nd region 33 are adjacent to each other and the phase values are continuous on the boundaries thereof. Thus, since the basic phase pattern 31 is single-purified, the sheet-like excitation light L1 can be generated with a simpler structure without using a complicated optical element or the like.

Since the light scanning section 13 for scanning the excitation light L1 with respect to the sample S is provided in the light sheet microscope 1, the excitation light L1 irradiated from the 1 st objective lens 16 can be scanned with respect to the sample S.

In the light sheet microscope 1, the photodetector 20 is a two-dimensional image pickup device having a plurality of pixel columns R and capable of rolling readout. This can improve S/N compared with the case of using a two-dimensional image pickup device that can be read out entirely.

The preferred embodiments of the present invention have been described above, but the present invention is not limited to the above embodiments. For example, the basic phase pattern 31 of modification 1 shown in fig. 5 may be used. In the 2 nd region 33A of the basic phase pattern 31A, the phase value decreases by 4 pi radians only straight along the direction D1. That is, the absolute value of the slope of the phase value in the 1 st region 32 is different from the absolute value of the slope in the 2 nd region 33A. The basic phase pattern 31A is non-line symmetrical about the center line C. In this example, the boundary between the 1 st region 32 and the 2 nd region 33A is also located on the center line C.

Even in the case where such a basic phase pattern 31A is used, the excitation light L1 in the form of a sheet is generated by modulating the excitation light L1 by the SLM14 in accordance with the basic phase pattern 31A as shown in fig. 6. In this case, as shown in fig. 6 (a), in the excitation light A2 incident on the 2 nd region 33A, the retardation amount of the phase on the SLM14 is larger than that of the excitation light A1 incident on the 1 st region 32. Also, in the excitation light B2, the amount of retardation of the phase on the SLM14 is larger than that of the excitation light B1, and in the excitation light C2, the amount of retardation of the phase on the SLM14 is larger than that of the excitation light C1. Thereby, the sheet-like excitation light L1 is generated at the imaging position Y2 farther from the 1 st objective lens 16 than the imaging position Y1 in the case of the above embodiment. The sheet-like excitation light L1 is generated at a position different from the optical axis X.

As described above, even in the modification 1, the sheet-like excitation light L1 can be irradiated from the 1 st objective lens 16 onto the sample S, and the sheet-like excitation light L1 can be generated with a simple configuration, as in the case of the embodiment described above. In modification 1, since the basic phase pattern 31 is non-linear symmetrical with respect to the straight line C, the sheet-like excitation light L1 can be generated at a position different from the optical axis X of the 1 st objective lens 16.

The basic phase pattern 31B of modification 2, the basic phase pattern 31C of modification 3, or the basic phase pattern 31D of modification 4 shown in fig. 7 may be used. In the 1 st region 33B of the basic phase pattern 31B, the phase value increases by 4 pi radians only straight along the direction D1. That is, the absolute value of the slope of the phase value in the 1 st region 32B and the absolute value of the slope of the phase value in the 2 nd region 33A are equal.

In the basic phase pattern 31C, the positional relationship in the direction D1 of the 1 st region 32 and the 2 nd region 33 is opposite to that in the above-described embodiment. I.e. the one that is the one. The phase value increases as the distance from the boundary (center line C) in the 1 st region 32 becomes smaller and the phase value decreases as the distance from the boundary in the 2 nd region 33 becomes smaller in the basic phase pattern 31C, and the phase value increases as the distance from the boundary in the 1 st region 32 becomes smaller and the phase value decreases as the distance from the boundary in the 2 nd region 33 becomes larger, relative to the above-described basic phase pattern 31.

In the basic phase pattern 31D, the positional relationship in the direction D1 of the 1 st region 32D and the 2 nd region 33D is opposite to that in the case of the above embodiment, as in the above modification 3. In the basic phase pattern 31D, the phase value increases by 4 pi radians only straight in the 1 st region 32D along the direction D1 and decreases by 4 pi radians only straight in the 2 nd region 33D along the direction D1. The basic phase patterns 31B to 31D are line-symmetrical with respect to the center line C. Even in the basic phase patterns 31B to 31D, the boundary between the 1 st region 32 and the 2 nd region 33A is located on the center line C.

Even when these basic phase patterns 31B to 31D are used, the sheet-like excitation light L1 can be irradiated from the 1 st objective lens 16 onto the sample S, and the sheet-like excitation light L1 can be generated with a simple configuration, as in the case of the above-described embodiment.

As shown in fig. 8, the phase pattern P may be calculated by superimposing the diffraction grating pattern 41 in the form of a diffraction grating on the basic phase pattern 31. The diffraction grating pattern 41 is formed in a diffraction grating shape in the direction D2. As shown in fig. 9, the phase pattern P may be calculated by superimposing a lenticular lens pattern 42 such as a fresnel lens on the basic phase pattern 31. The phase pattern P may be calculated by superimposing the diffraction grating pattern 41 or the lens pattern 42 on the basic phase patterns 31A to 31C.

Even in these cases, as in the case of the above embodiment, the sheet-like excitation light L1 can be irradiated from the 1 st objective lens 16 onto the sample 16, and the sheet-like excitation light L1 can be generated with a simple structure. The phase of the excitation light can be set to be diffraction grating-like or lens-like without providing a diffraction grating or lens element. Therefore, the sheet-like excitation light L1 can be generated with a simpler structure.

The light sheet microscope 1D according to modification 5 shown in fig. 10 may be configured as well. The light sheet microscope 1D is different from the light sheet microscope 1 of the above embodiment in that the light sheet microscope 1D includes a1 st optical system 15D constituting a telescope optical system. The 1 st optical system 15D has 2 lenses 15da,15db for condensing the excitation light L1 from the SLM14 at the pupil of the 1 st objective lens 16, and constitutes a telescope optical system. Examples of such telescope optical systems include a 4f optical system, a kepler optical system, and a galilean optical system. The 1 st optical system 15D and the 1 st objective lens 16 constitute an irradiation optical system.

Even by such a light sheet microscope 1D, the excitation light L1 is modulated with the SLM14 in accordance with the basic phase pattern 31 as shown in fig. 11, thereby generating the excitation light L1 in a sheet shape. As described above, even in the modification 5, the sheet-like excitation light L1 can be irradiated from the 1 st objective lens 16 onto the sample S, and the sheet-like excitation light L1 can be generated with a simple configuration, as in the case of the embodiment described above.

The basic phase pattern 31 of the above embodiment may be further provided in the 3 rd region where the phase value in the direction D1 is constant. Such a 3 rd region is provided between, for example, the 1 st region 32 and the 2 nd region 33. In this case, the 1 st region 32 and the 2 nd region 33 are not adjacent to each other.

In the above embodiment, the photodetector 20 may be a rolling-readout-enabled area image sensor (area image sensor). The 1 st optical system 15 may be omitted, and the excitation light L1 output from the SLM14 may be directly input to the 1 st objective lens 16. The light scanning section 13 may be configured to receive the excitation light L1 output from the SLM14. The optical axis of the 1 st objective lens 16 and the optical axis of the 2 nd objective lens 17 may not be orthogonal or may not intersect each other.

The control unit 21 may control the wavelength of the generated sheet-like excitation light L1, the thickness of the sheet, the light-collecting position, the shape, or the like by appropriately changing the phase pattern P. The control unit 21 may superimpose a pattern for aberration correction on the basic phase pattern 31 and calculate the phase pattern P. The pattern for aberration correction may be created based on the image data output from the photodetector 20. This enables feedback correction. For example, an optical element having a slit for blocking the 0 th order light of the excitation light L1 or Gao Ciguang may be provided between the lens 15a and the 1 st objective lens 16. This makes it possible to shield unnecessary light (light that may become noise).

The control unit 21 may set the number of pixel columns N to be simultaneously exposed on the photodetector 20 capable of rolling readout in accordance with the phase pattern P. At this time, the control unit 21 sets a readout period T2 of the rolling readout based on the set pixel column number N and the exposure period T1 of each pixel column. In this case, the exposure period T1 of each pixel row is set by a user or the like. The control unit 21 may set the exposure period T1 for each pixel column based on the set pixel column number N and the readout period T2 for the rolling readout. In this case, the readout period T2 of the scroll readout is set by a user or the like. In either case, the control unit 21 controls the optical scanning unit 13 or the photodetector 20 so that scanning by the excitation light L1 of the optical scanning unit 13 and signal reading by the pixel rows R of the photodetector 20 that can be read by rolling are synchronized.

The irradiation optical system and the detection optical system may not include an objective lens. In this case, a condenser lens may be used instead of the 1 st objective lens 16 or the 2 nd objective lens 17.

Description of symbols

A1 … light sheet microscope (image acquisition device), an 11 … light source, a 13 … light scanning section, a 14 … spatial light modulator, a 16 … 1 st objective lens, a 17 … 2 nd objective lens, a 20 … light detector, a 21 … control section, a 22 … computer, a 23 … controller, a 31 … basic phase pattern, a 32 … 1 st area, a 33 … 2 nd area, a 41 … diffraction grating pattern, a 42 … lens pattern, a C … straight line (center line), a D1 … prescribed direction, L1 … excitation light, L2 … detection light, a P … phase pattern, an R … pixel array, and a S … sample.

Claims

1. An image acquisition apparatus, characterized in that:

the device is provided with:

a light source that outputs excitation light including a wavelength at which a sample is excited;

a spatial light modulator having a plurality of pixels arranged in two dimensions and modulating a phase of the excitation light for each of the plurality of pixels;

an irradiation optical system including an objective lens for irradiating the modulated excitation light to a sample;

a detection optical system that images detection light emitted from the sample in association with irradiation of the excitation light from the irradiation optical system;

a photodetector that captures an image of the detection light imaged by the detection optical system; and

a control unit that controls the amount of phase modulation for each of the plurality of pixels in accordance with a phase pattern in which phase values corresponding to the plurality of pixels are distributed two-dimensionally,

the phase pattern is a phase pattern generated based on a prescribed basic phase pattern,

the basic phase pattern has a1 st region in which the phase value continuously increases in a prescribed direction, and a2 nd region which is opposite to the 1 st region in the prescribed direction and in which the phase value continuously decreases in the prescribed direction,

the basic phase pattern is non-line symmetrical with respect to a center line passing through a center in the predetermined direction and orthogonal to the predetermined direction so that an absolute value of a slope of the phase value in the 1 st region and an absolute value of a slope of the phase value in the 2 nd region are different, and a boundary between the 1 st region and the 2 nd region is located on the center line, whereby the excitation light of a sheet shape is generated at a position different from an optical axis of the objective lens.

2. The image acquisition apparatus according to claim 1, wherein:

in the 1 st region, the phase value increases linearly along the predetermined direction, and in the 2 nd region, the phase value decreases linearly along the predetermined direction.

3. The image acquisition apparatus according to claim 1 or 2, wherein:

the 1 st region and the 2 nd region are adjacent to each other and the phase value is continuous on the boundary thereof.

4. The image acquisition apparatus according to any one of claims 1 to 3, wherein:

the phase pattern is a phase pattern in which a diffraction grating pattern in the form of a diffraction grating and the basic phase pattern are superimposed.

5. The image acquisition apparatus according to any one of claims 1 to 4, wherein:

the phase pattern is a phase pattern in which a lenticular lens pattern and the basic phase pattern are overlapped.

6. The image acquisition apparatus according to any one of claims 1 to 5, wherein:

the sample analyzer further comprises an optical scanning unit for scanning the excitation light with respect to the sample.

7. The image acquisition apparatus according to any one of claims 1 to 6, wherein:

the photodetector is a region image sensor having a plurality of pixel columns and capable of rolling readout.

8. The image acquisition apparatus according to any one of claims 1 to 7, wherein:

the image acquisition device is a light sheet microscope.

9. An image acquisition method, characterized in that:

comprising:

a modulating step of modulating, by a spatial light modulator having a plurality of pixels arranged in two dimensions, a phase of excitation light including a wavelength at which a sample is excited for each of the plurality of pixels;

an irradiation step of irradiating the sample with the modulated excitation light by an objective lens; and

an imaging step of imaging detection light emitted from the sample in association with the irradiation of the excitation light and imaging an image of the imaged detection light,

in the modulating step, the phase modulation amount of each of the plurality of pixels is controlled in accordance with a phase pattern which is generated based on a prescribed basic phase pattern and which is two-dimensionally distributed corresponding to the phase values of the plurality of pixels, respectively,